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Comparable respiratory activity in attached and suspended human fibroblasts.
Zdrazilova, Lucie; Hansikova, Hana; Gnaiger, Erich.
Afiliação
  • Zdrazilova L; Department of Pediatrics and Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General Hospital in Prague, Prague, Czechia.
  • Hansikova H; Department of Pediatrics and Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General Hospital in Prague, Prague, Czechia.
  • Gnaiger E; Oroboros Instruments, Innsbruck, Austria.
PLoS One ; 17(3): e0264496, 2022.
Article em En | MEDLINE | ID: mdl-35239701
ABSTRACT
Measurement of oxygen consumption of cultured cells is widely used for diagnosis of mitochondrial diseases, drug testing, biotechnology, and toxicology. Fibroblasts are cultured in monolayers, but physiological measurements are carried out in suspended or attached cells. We address the question whether respiration differs in attached versus suspended cells using multiwell respirometry (Agilent Seahorse XF24) and high-resolution respirometry (Oroboros O2k), respectively. Respiration of human dermal fibroblasts measured in culture medium was baseline-corrected for residual oxygen consumption and expressed as oxygen flow per cell. No differences were observed between attached and suspended cells in ROUTINE respiration of living cells and LEAK respiration obtained after inhibition of ATP synthase by oligomycin. The electron transfer capacity was higher in the O2k than in the XF24. This could be explained by a limitation to two uncoupler titrations in the XF24 which led to an underestimation compared to multiple titration steps in the O2k. A quantitative evaluation of respiration measured via different platforms revealed that short-term suspension of fibroblasts did not affect respiratory activity and coupling control. Evaluation of results obtained by different platforms provides a test for reproducibility beyond repeatability. Repeatability and reproducibility are required for building a validated respirometric database.
Assuntos

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Fosforilação Oxidativa / Respiração Celular Limite: Humans Idioma: En Ano de publicação: 2022 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Fosforilação Oxidativa / Respiração Celular Limite: Humans Idioma: En Ano de publicação: 2022 Tipo de documento: Article